Project Summary: A major goal of cell biology is to understand the structures, interactions, functions and regulation of cellular organelles. Today, Nobel-worthy research of Betzig, Hell, Moerner, Zhuang, and now many others allows scientists to visualize organelles in live cells at resolutions that number in the tens of nanometers. The problem is that the images last for only seconds, and thus the pictures, however beautiful, hide complex dynamics and interactions that underlie organelle function in health and disease. This proposal expands a toolkit of two-component chemical tools called ?HIDE? (High Density Environment-Sensitive) probes that will allow scientists to visualize the dynamics of multiple organelles, simultaneously, at super-resolution, for unprecedented times, and in multiple colors. HIDE probes consist of two parts, an organelle- specific membrane probe with a reactive functional group (such as trans-cyclooctene (TCO)) and a silicon rhodamine dye (SiR or, in this proposal, HMSiR) with a reaction partner (such as tetrazine). These two parts, when added sequentially to live cells, undergo an in situ tetrazine ligation reaction that localizes the SiR dye at high density within the organelle membrane. Here HMSiR photostability is significantly enhanced and can generate single molecule switching (SMS) nanoscopy images up to 50 times longer than HMSiR-tagged proteins. The first HIDE probes (used to image the Golgi, ER, mitochondria, and plasma membrane) have been reported by us. Here we build on this strong foundation and expand the HIDE toolbox to include probes for four additional organelles (early, late, and recycling endosomes, and cilia) (Aim 1), coupled with orthogonal SMS dyes and chemistries (Aim 2) required for two- and eventually three-color long-term SMS nanoscopy. In the final Aim (Aim 3), we develop HIDE-EM probes to highlight discrete organelles in 3D by electron microscopy (EM). Each Aim is associated with deliverables and time-dependent milestones and is supported by significant preliminary results. Although we focus here on developing HIDE probes useful for SMS microscopy, only minor adjustments in the dye identity would be needed to generate HIDE probes for STED. The technology developed herein will greatly enhance the power, scope, and impact of long time-lapse live cell nanoscopy by expanding the number and types of organelles that can be evaluated, alone, in combination using multiple colors, using different imaging modalities, and in both cultured and hard-to-transfect primary cells.